In order to maximize the imaging penetration depth into tissue using multiphoton microscopy, the nonlinear optical (NLO) properties of the molecular probes developed at Wash U will be characterized in the near infrared (NIR) spectral region. The properties relevant to in vivo imaging include the two-photon absorption cross-section, fluorescence yield, and net two photon yield as a function of wavelength. This information will be iteratively fed back to optimize the NLO properties for excitation in the NIR, as well as to develop an instrument that complements the contrast agents with the nominal biochemical properties (e.g., tumor affinity, specificity, etc.) The NLO properties of the dyes will dictate the specifications of a multiphoton excitation fluorescence microscope operating in the NIR, where the attenuation coefficient in tissue is lower than in the visible range, where fluorescence microscopy is typically performed. Such an instrument will motivate further development of clinical and endoscopic diagnostic imaging systems for in vivo molecular imaging of cancer. The study will be guided by three Specific Aims: Aim I: Synthesize novel NIR dyes for 2-multiphoton microscopy. Peptides targeting integrins will be labeled with the new dyes to improve selectivity for imaging tumor cells and tissues. Aim II: Develop a platform for characterizing the nonlinear optical properties of the dyes Aim III: Develop fiber-based NIR multiphoton microscope AIM IV: Evaluate the detection/imaging of orthotopic tumors in mice by the use of the multiphoton microscope and tumor-specific molecular probes. Successful completion of this project will result in the development of a novel NIR multiphoton microscope and molecular probes for cellular and tissue imaging of cancers by optical methods. Translation of the method and instrument developed from small animals to imaging of human Gl and cervical cancers is expected at the completion of the study.